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Previous Article | Next Article 
Journal of Bacteriology, November 2000, p. 6259-6263, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification by Gene Deletion Analysis of a Regulator, VmsR,
That Controls Virginiamycin Biosynthesis in Streptomyces
virginiae
Ryu
Kawachi,
Usamas
Wangchaisoonthorn,
Takuya
Nihira,* and
Yasuhiro
Yamada
Department of Biotechnology, Graduate School
of Engineering, Osaka University, Osaka 565-0871, Japan
Received 13 March 2000/Accepted 9 August 2000
 |
ABSTRACT |
Virginiae butanolide (VB)-BarA of Streptomyces
virginiae is one of the newly discovered pairs of a butyrolactone
autoregulator and a corresponding receptor protein of
Streptomyces species and regulates the production of the
antibiotic virginiamycin (VM) in S. virginiae. The gene
vmsR was found to be situated 4.7 kbp upstream of the
barA gene, which encodes the VB-specific receptor. The
vmsR product was predicted to be a regulator of VM
biosynthesis based on its high homology to some
Streptomyces pathway-specific transcriptional regulators
for the biosynthetic gene clusters of polyketide antibiotics, such as
Streptomyces peucetius DnrI (47.5% identity, 84.3%
similarity), which controls daunorubicin biosynthesis. A
vmsR deletion mutant was created by homologous recombination. Neither virginiamycin M1 nor virginiamycin S
was produced in the vmsR mutant, while amounts of VB and
BarA similar to those produced in the wild-type strain were detected.
Reverse transcription-PCR analyses confirmed that the vmsR
deletion had no deleterious effects on the transcription of the
vmsR-surrounding genes, indicating that VmsR is a positive
regulator of VM biosynthesis in S. virginiae.
| |
TEXT |
Streptomycetes are gram-positive
filamentous bacteria that are well-known for producing a vast array of
bioactive compounds, including more than 70% of commercially important
antibiotics. The production of antibiotics by these organisms is
regulated by a variety of physiological and nutritional conditions and
is coordinated with processes of morphological differentiation, such as
the formation of aerial mycelia and spores. Despite many years of
research on antibiotics driven by their commercial importance, the
overall regulatory pathway governing antibiotic production is still
poorly understood. A detailed knowledge of the signal cascade and the
genetic components involved in antibiotic production should permit
construction of strains that can overproduce these commercially
important compounds.
Antibiotic production and/or morphological differentiation is
controlled in several Streptomyces species by
low-molecular-weight compounds called butyrolactone autoregulators
(32). To date, 11 butyrolactone autoregulators have been
chemically identified and classified into three types based on minor
structural differences in their C-2 side chains: (i) the virginiae
butanolide (VB) type, containing a 6-
-hydroxy group (13,
31); (ii) the IM-2 type, containing a 6-
-hydroxy group
(25, 27); and (iii) the A-factor type, containing a 6-keto
group (16). Their effectiveness at nanomolar concentrations,
as well as the presence in these species of specific receptor proteins
(BarA as a VB-specific receptor in Streptomyces virginiae
[9, 10, 21], FarA as an IM-2-specific receptor in
Streptomyces lavendulae FRI-5 [23, 28], and
ArpA as an A-factor-specific receptor in Streptomyces
griseus [22]) as mediators of autoregulator
signaling, implies that they should be regarded as
Streptomyces hormones.
VB-BarA of S. virginiae has been among the most frequently
studied pairs and is known to regulate the coordinated production of
two structurally different compounds, virginiamycin M1
(VM1) and virginiamycin S (VS), a pair of antibiotics
showing strong synergistic bactericidal activity. In vitro (11,
12) and in vivo (11, 17, 18) analyses have
demonstrated that BarA in the absence of VB is a DNA-binding
transcriptional repressor and that the release of DNA-bound BarA in the
presence of VB from the promoter region of a target gene(s) will
result, ultimately, in virginiamycin (VM) production. However, the VB
signaling cascade beyond BarA is not clear at present.
In our previous study on a pleiotropic regulatory gene
(barX) situated 259 bp upstream of the barA gene
(8), a phenotypic defect in VM and VB production in the
barX deletion mutant was correlated with the lack of
transcription of a plausible pathway-specific regulatory gene
(vmsR; formerly called orf2), which raised the possibility that the vmsR product might be responsible for
the initiation of VM and/or VB production. To clarify the in vivo function of the VmsR protein, we created a vmsR deletion
mutant of S. virginiae by homologous recombination and
report here the results from phenotypic and transcriptional analyses on
the vmsR mutant, which clearly indicate that VmsR is the
regulator positively controlling the biosynthesis of both
VM1 and VS in S. virginiae.
Bacterial strains, plasmids, growth conditions, and transformation.
S. virginiae (strain MAFF 10-06014; National Food Research
Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Japan) was grown at 28°C in modified yeast extract-malt extract liquid medium (7) for protoplast formation, in tryptic soy broth (Oxoid, Basingstoke, Hampshire, United Kingdom) containing thiostrepton (5 µg ml
1) for plasmid preparation, on
ISP2 agar medium (Difco, Detroit, Mich.) for spore formation, and in
liquid f medium (33) for VM and VB production. S. virginiae was transformed as described previously (7).
A plasmid, pSVR1, containing a 3.5-kbp
EcoRI-BamHI fragment carrying
barZ-vmsR-varM (8) (DDBJ accession no. AB035547) in the EcoRI-BamHI site of pUC18 was used as a
template for PCR. DNA manipulations in Escherichia coli and
S. virginiae were performed as described by Sambrook et al.
(24) and Hopwood et al. (6), respectively.
Construction and identification of a vmsR deletion
mutant.
The vmsR gene product was predicted to be a
regulator of VM biosynthesis based on its high homology to some
Streptomyces pathway-specific transcriptional regulators of
the biosynthetic gene clusters of polyketide antibiotics, such as
Streptomyces peucetius DnrI (47.5% identity, 84.3%
similarity), which controls daunorubicin biosynthesis (26);
Streptomyces coelicolor A3(2) ActII-ORF4 (36.1% identity, 76.6% similarity), which controls actinorhodin biosynthesis
(3); and S. coelicolor A3(2) RedD (28.9%
identity, 67.3% similarity), which controls undecylprodigiosin
biosynthesis (19). Alternatively, the VmsR protein might be
responsible for the initiation of VB biosynthesis, because the
phenotypic defect of VB production in the barX mutant
correlated with the lack of vmsR transcription (8). In order to investigate these possibilities, a
vmsR deletion mutant was created by homologous recombination
within the S. virginiae genome (Fig.
1A).
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FIG. 1.
Replacement of the S. virginiae vmsR gene
with plasmid pSVR301 to form truncated vmsR by homologous
recombination. (A) Restriction maps of pSVR301 and the genomic DNA of
the S. virginiae wild-type strain. The varM
promoter region was amplified by PCR with primers
5'-GGGGCCACTCGCGACTCCAACCGCCGC-3' (a newly
created recognition site for NruI is indicated by
underlining) and 5'-GCAAGCGGCTCGCCGTACGGGAGCGCTG-3'
(a BsiWI recognition site is indicated by underlining)
using pSVR1 as a template. The
NruI-BsiWI-digested PCR fragment was then used to
replace the original 1.0-kbp NruI-BsiWI fragment
of pSVR1, resulting in the deletion of a 145-bp 5' untranslated region
and the N-terminal 698 bp of vmsR. From the resulting
plasmid (pSVR201), a 2.8-kbp EcoRI-HindIII
fragment containing barZ, the C-terminal 118 bp of
vmsR, and the entire varM gene was cloned into
the EcoRI-HindIII site of pUWL-KS to create
plasmid pSVR301. A single crossover between pSVR301 and a homologous
DNA in the genome gave the pSVR301-integrated strain, and a second
crossover generated a vmsR-disrupted strain. Only one of the
two possible first crossover events is shown. The hatched box indicates
the region deleted by the second crossover. (B) Hybridization patterns
of BamHI-digested genomic DNA of the pSVR301-integrated
strain (lane 1), vmsR-disrupted strain (lane 2), wild-type
segregant (lane 3), and the S. virginiae wild-type strain
(lane 4). A 2.8-kbp BamHI fragment containing the
vmsR gene was used as a probe. When hybridized with the
probe, the vmsR-disrupted strain (lane 2) gave a 2.0-kbp
fragment, as a result of the deletion of the vmsR gene.
|
|
Plasmid pSVR301 was constructed by ligating a 2.8-kbp
EcoRI-HindIII fragment containing
barZ-
vmsR-varM into the
EcoRI-HindIII site of pUWL-KS
(29). The S. virginiae wild-type strain was transformed with pSVR301, and a single crossover between pSVR301 and a
homologous DNA in the S. virginiae genome gave the
pSVR301-integrated strain. When we cultivated the pSVR301-integrated
strain in liquid tryptic soy broth in the absence of thiostrepton, we
obtained two types of strains: one is a vmsR deletion
mutant, and the other is a regenerated wild-type strain (Fig. 1B). The
integration of pSVR301 in the first crossover strain and its deletion
in the second crossover strain were confirmed by Southern blot
hybridization using a 2.8-kbp BamHI fragment encoding
vmsR as a probe (Fig. 1B). We also confirmed the 802-bp
deletion in the vmsR mutant by DNA sequence analysis. One of
the resulting vmsR deletion mutants (strain DR1) was used
for further investigation.
Phenotypes of the vmsR deletion mutant.
The
amounts of VM produced were determined by reverse-phase high-pressure
liquid chromatography (HPLC) using purified VM1 and VS as
standards under the following conditions: column, Cosmosil 5C18 (4.6 by 100 mm; Nacalai Tesque, Kyoto, Japan); flow
rate, 0.75 ml/min; temperature, 40°C; detection, UV radiation at 305 nm; elution, 5 min with 20% CH3CN containing 0.1%
trifluoroacetic acid followed by the linear increase of
CH3CN from 20 to 80% for 15 min. The amounts of VB were
determined by measuring the VB-dependent production of VM
(20). VB binding activity was assayed by the ammonium
sulfate precipitation method (10) with
3H-labeled VB-C7 (54.6 Ci/mmol) in the presence
and absence of 2,000-fold cold VB-C. When traits relating to VB-BarA in
S. virginiae were measured (Table
1), all of them (VB production, VB
binding activity, VM production, VM resistance, and morphology) were
found to be identical, with high reproducibility, in the wild-type
strain and the wild-type segregant (Table 1), indicating that no
unexpected mutations relating to the VB-BarA signal transduction
pathway took place during the pSVR301 integration into the wild-type
strain or during the second crossover event generating either the
wild-type segregant or the vmsR disruptant. The
vmsR mutant, however, did not produce any VM, even after
48 h of cultivation, and was slightly more sensitive to
VM1 and VS (Table 1). No differences in VB production, VB
binding activity, growth rate, or morphology on either solid or liquid
media were observed between the vmsR mutant and the
wild-type strain (Table 1). The phenotypic defect of VM production in
the vmsR mutant was complemented by introducing intact
vmsR on pUWL-KS into the vmsR mutant (data not
shown). These results suggested that VmsR does not participate in
morphological control or VB biosynthesis in S. virginiae but
does participate, directly or indirectly, in the regulation of VM
biosynthesis.
Analysis of antibiotic products.
A further experiment was
designed to determine the amounts of two components of VM, namely
VM1 and VS, because the loss of either VM1 or
VS production in the vmsR mutant would lead to great loss of
antibiotic activity from the synergistic action of VM1 and
VS. The 24-h culture supernatant was recovered after centrifugation, and the amounts of VM1 and VS were analyzed by HPLC. Under
the HPLC conditions employed, VM1 and VS produced by the
wild-type strain were detected easily at retention times of
approximately 18 and 21 min, respectively (Fig.
2A), while the vmsR mutant did not show any signs of VM1 or VS (Fig. 2B), indicating
clearly that the VmsR protein is essential for the production of both VM1 and VS.
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FIG. 2.
Detection of two components of antibiotics,
VM1 and VS, by C18 reverse-phase HPLC analysis.
Authentic VM1 and VS were purified from the commercial
animal-feed additive STAFAC 500 (Smith Kline-RIT, Rixensart, Belgium)
by C18 reverse-phase HPLC (14).
|
|
Transcriptional patterns of the vmsR-surrounding genes
in the vmsR mutant and the wild-type strain.
Reverse
transcription (RT)-PCR of two genes immediately adjacent to
vmsR, namely barZ transcribed in the same
direction as vmsR and varM transcribed
divergently from vmsR, revealed that transcription of these
genes in the vmsR mutant is similar to that in the wild-type
strain, indicating that the deletion in vmsR did not have a
polar effect on barZ or a deleterious effect on
varM. Therefore, the defect in VM biosynthesis in the
vmsR mutant can be concluded to be due to the lack of
functional VmsR protein rather than any undesired effect on the
surrounding genes. Thus far, six other genes (orf4,
orf5, barX, barA, barB, and
varS) have been sequenced, and none of them showed any
changes in transcription in the vmsR deletion mutant (Fig.
3), which suggests that they are not
under the control of vmsR.
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FIG. 3.
(A) Gene organization in the 10-kbp region containing
the vmsR gene in S. virginiae. Solid arrows, gray
arrows, and open arrows indicate regulator genes, resistance genes for
VM, and genes of unknown function, respectively. (B) Transcriptional
comparison of vmsR-surrounding genes in the vmsR
disruptant and the wild-type strain by RT-PCR. RT-PCR conditions were
previously described (8). Production of VB and
VM1 plus VS is indicated by open and solid arrows,
respectively. With the entire sample, RT-PCR was carried out without
reverse transcriptase to confirm that the signals shown were derived
from mRNA and not from contaminating genomic DNA. vmsR,
vmsR disruptant; Wild-type, S. virginiae MAFF
10-06014.
|
|
Based on the phenotypic analysis, the level of resistance to
VM1 and VS was slightly reduced in the vmsR
mutant compared to that in the wild-type strain (Table 1), although the
transcription of the resistance genes (varS, which encodes
the VS transporter and therefore confers VS resistance
[14], and varM, which likely encodes the
VM1 transporter [8]) was not impaired in
the vmsR mutant (Fig. 3). In the case of varS, in
addition to the VB-dependent derepression of barB-varS
bicistronic transcription, varS monocistronic transcription
was induced by the presence of VS (14), which should
function to strengthen the VS resistance level. Similar regulation to
enhance the VM1 resistance may be operative in the presence
of VM1; the absence of VM1 and VS (i.e., not
the absence of the VmsR protein per se) in the vmsR mutant
seems to be the main reason for the slightly lowered resistance.
The amino acid sequence of VmsR is very similar to those of DnrI,
ActII-ORF4, and RedD, all of which belong to an expanding family of
Streptomyces antibiotic regulatory proteins (SARPs). SARPs
are predicted to have a similar mechanism of transcriptional activation
through binding to specific nucleotide sequences, and probable DNA
recognition sites (helix 3 and a loop connecting two C-terminal
-strands [6 and 7]) have been estimated on the basis of the
crystal structure of OmpR (30). Alignment of the DNA-binding domains of OmpR with homologous regions of VmsR and SARPs
reveals a number of highly conserved amino acids. Recently, as
predicted by Wietzorrek and Bibb (30), the ActII-ORF4
protein has been confirmed to be a DNA-binding transcriptional
activator by gel shift assays and DNase I footprinting (1).
Thus, it seems highly probable that VmsR is a DNA-binding protein which acts as a transcriptional activator. It is currently unclear whether VmsR activates VM production by directly activating the transcription of the biosynthetic gene cluster for VM or via another regulator, because no biosynthetic genes for VM have been cloned from S. virginiae, although biosynthetic genes for the closely related antibiotic pristinamycin have been found in Streptomyces
pristinaespiralis (2).
In this work, we obtained in vivo evidence that VmsR is a positive
regulator of the biosynthesis of both VM1 and VS by
constructing and analyzing the vmsR deletion mutant.
However, it is not clear at present whether the transcription of
vmsR is regulated directly by VB-BarA or indirectly via
another regulator(s), such as the BarB protein. We are currently
constructing a barB disruptant, the phenotypic and
transcriptional analyses of which will clarify the transcriptional
cascades among several regulators, such as BarA, VmsR, and BarB.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the Research for
the Future Program of the Japan Society for the Promotion of Science (JSPS).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7433. Fax:
81-6-6879-7432. E-mail:
nihira{at}biochem.bio.eng.osaka-u.ac.jp.
| |
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Journal of Bacteriology, November 2000, p. 6259-6263, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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